Active transport of required metabolites in gram negative organisms is the result of complex interactions between cellular constituents localized in the outer and cytoplasmic membranes and the periplasmic space. The overall objectives of our research plan are to develop a model transport system in which these interactions may be probed. In particular, this proposal is designed to delineate structural and functional features of three specific Escherichia coli proteins involved in the biosynthesis (EntD) of the catechol siderophore enterobactin (enterochelin) and the siderophore-mediated uptake (FepA, Fes) of the nutritionally important trace element, iron. The structural genes for these three components have been isolated and their nucleotide sequences determined. The protein products have been identified and in come cases purified. In vitro and in vivo mutagenesis procedures along with protein biochemistry protocols will be employed to evaluate structural and functional features of: (1) the outer membrane receptor (FebA) binding sites for its three specific ligands, ferric enterobactin, colicin D and colicin B; (2) the cytoplasmic enzyme Fes, which is responsible for removing iron from the siderophore complex upon its entry into the cell; and (3) the membrane-associated biosynthetic enzyme EntD, including the nature of its molecular association with the other components (EntE, EntF and EntG) of the multienzyme complex enterobactin synthetase, and its potential role in extracellular release of newly synthesized siderophore molecules. Aside from its basic scientific interest as a model high affinity transport system, an understanding of the mechanisms of iron transport is significant is that in all organisms studied, from bacterial to man, the metabolic iron state of the organism regulates the degree of nutritive iron absorption and iron has been documented as an important factor of the pathophysiology of disseminating gram negative infections in man and animals. It is hoped that this system may present a model defining the essential characteristics of iron transport for comparison with similar systems from other gram negative pathogens and against which effective future therapies may be targeted.